Structure, Mechanism and Regulation of the V-ATPases

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The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V- ATPases). The V-ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, pH homeostasis, bone resorption and tumor metastasis. Understanding how V-ATPases are regulated is thus crucial to understanding many disease processes, including viral entry, osteoporosis and metastasis. The V-ATPases are organized into two functional domains: a peripheral V1 domain responsible for ATP hydrolysis and a integral V0 domain responsible for proton translocation. Electron microscopic images of the V-ATPase complex reveal multiple connections between the V1 and V0 domains. To determine the arrangement of subunits within the V-ATPase complex, unique cysteine residues will be introduced into the B subunit and used as sites of attachment of a photoactivated crosslinker. In addition, electron microscopy of complexes decorated with subunit- specific antibodies will be performed. The function of a unique domain of the catalytic A subunit will be addressed by deletion and random mutagenesis. The structure of the 100 kDa a subunit and its interactions with the proteolipid subunits of the V0 domain will be determined using cysteine mutagenesis, chemical labeling and disulfide bond formation. Finally, the in vivo dissociation of the V-ATPase complex, which has been proposed to be an important regulatory mechanism, will be investigated. Dissociation in response to glucose depletion will be compared in V-ATPases located in different intracellular compartments and mutants defective in dissociation will be selected and analyzed. These studies should provide further insight into the structure and regulation of this important family of (H+)-ATPases.